Development and application of non-invasive imaging techniques to physically and functionally assess the potential of novel cardiomyocyte stem cells to regenerate damaged myocardium have clear clinical significance. Repair and replacement of damaged cardiac muscle tissue is a key limitation in the recovery from ischemic and other forms of heart disease, the leading cause of death in the United States. New strategies for regenerating damaged cardiac muscle are urgently needed and could be readily translated to the clinical setting if first shown to be effective in animal models. The proposal outlined here will demonstrate the feasibility of using molecular imaging techniques in vivo to evaluate our novel stem cell-based strategy for cardiac regenerative medicine. This research proposal directly addresses both of the stated goals of the initiative outlined in RFA-HL-04-003 (Cellular and Molecular Imaging of the Cardiovascular, Pulmonary, and Hematopoetic Systems): (1) Detect and quantify molecular and cellular pathways that regulate heart function...(we describe and will further characterize a novel cardiomyocyte differentiation pathway), and (2) Develop new methods for cell tracking in vivo for applications in cell-based therapeutics...(we will transplant novel cardiomyocyte stem cells into damaged hearts and track them in vivo using advanced magnetic resonance and bioluminescent imaging methods). Our data indicate that cardiac cells transiently expressing the enzyme, Phenylethanolamine n-methyltransferase (Pnmt), ultimately become myocytes that contribute substantially to pacemaking, conduction, and working myocardium. Thus, Pnmt serves as a novel marker of cardiomyocyte stem cells (Pnmt+ cells) in the developing heart. We will test the idea that Pnmt+ cells can be selectively isolated and transplanted into damaged cardiac regions where they will be monitored both physically (location) and physiologically (function) using non-invasive molecular and cellular imaging techniques to evaluate the potential of these cells to regenerate cardiac muscle tissue in vivo. Three specific aims are proposed to accomplish this goal: Aim 1: Isolate and characterize Pnmt-nEGFP+ cells from cardiac-differentiated embryonic stem cells. Aim 2: In vivo magnetic resonance imaging (MRI) of transplanted cardiomyocyte stem (Pnmt+) cells. Aim 3: In vivo bioluminescence imaging (BLI) of transplanted cardiomyocyte stem (Pnmt+) cells. (End of Abstract)